Methods and materials for producing 5 and 7-carbon monomers

Abstract

This document describes biochemical pathways for biosynthesizing a 3-oxo-7-hydroxyheptanoyl-CoA intermediate using a β-ketothiolase, and enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoic acid. -7-hydroxyheptanoic acid can be further enzymatically converted to pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine or 1,7-heptanediol. This document also describes recombinant hosts producing 7-hydroxyheptanoic acid as well as pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine and 1,7-heptanediol.

1 . A method of producing 3-oxo-7-hydroxyheptanoyl-CoA or a salt thereof, said method comprising enzymatically converting 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA using a polypeptide having the activity of a β-ketothiolase classified under EC. 2.3.1.-. 
     
     
         2 . The method of  claim 1 , wherein said β-ketothiolase is classified under EC 2.3.1.16. 
     
     
         3 . The method of  claim 1 , wherein said β-ketothiolase is classified under EC 2.3.1.174. 
     
     
         4 . The method of  claim 1 , wherein said β-ketothiolase (a) has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 13 or (b) has at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO: 13 and is capable of converting 5-hydroxypentanoyl-CoA to 3-oxo-7-hydroxyheptanoyl-CoA. 
     
     
         5 . The method of  claim 1 , further comprising enzymatically converting 3-oxo-7-hydroxyheptanoyl-CoA to 7-hydroxyheptanoate using a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, a trans-2-enoyl-CoA reductase, and a thioesterase or a CoA transferase. 
     
     
         6 . The method of  claim 5 , wherein said 3-hydroxyacyl-CoA dehydrogenase or said 3-oxoacyl-CoA reductase is classified under EC 1.1.1.35, EC 1.1.1.36, EC 1.1.1.100, or EC 1.1.1.157. 
     
     
         7 . The method of  claim 5 , wherein said enoyl-CoA hydratase is classified under EC 4.2.1.17 or EC 4.2.1.119. 
     
     
         8 . The method of  claim 5 , wherein said trans-2-enoyl-CoA reductase is classified under EC 1.3.1.38, EC 1.3.1.44, or EC 1.3.1.8. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 5 , said method further comprising enzymatically converting 7-hydroxyheptanoate to pimelic acid, 7-aminoheptanoate, heptamethylenediamine, or 1,7-heptanediol in one or more steps. 
     
     
         12 . The method of  claim 11 , wherein 7-hydroxyheptanoate is converted to pimelic acid using one or more of a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxyvalerate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase. 
     
     
         13 . The method of  claim 11 , wherein 7-hydroxyheptanoate is converted to 7-aminoheptanoate using one or more of a polypeptide having the activity of an alcohol dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, and a ω-transaminase. 
     
     
         14 . The method of  claim 11 , wherein 7-hydroxyheptanoate is converted to heptamethylenediamine using one or more of a carboxylate reductase, a ω-transaminase, an alcohol dehydrogenase, an N-acetyltransferase, and an acetylputrescine deacylase. 
     
     
         15 . The method of  claim 13 , wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12. 
     
     
         16 . The method of  claim 11 , wherein 7-hydroxyheptanoate is converted to 1,7-heptanediol using a carboxylate reductase and an alcohol dehydrogenase. 
     
     
         17 . The method of  claim 16 , wherein said carboxylate reductase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 2-6. 
     
     
         18 . The method of  claim 1 , wherein said 5-hydroxypentanoyl-CoA is enzymatically produced from 2-oxoadipate or malonyl-CoA. 
     
     
         19 . The method of  claim 18 , wherein 5-hydroxypentanoyl-CoA is enzymatically produced from 2-oxoadipate—using one or more of an alpha-aminotransaminase; a 2-oxoadipate decarboxylase; a branch chain decarboxylase; a glutamate decarboxylase; a ω-transaminase; a CoA transferase, a CoA ligase, and an alcohol dehydrogenase. 
     
     
         20 . The method of  claim 18 , wherein 5-hydroxypentanoyl-CoA is enzymatically produced from malonyl-CoA using one or more of a malonyl-CoA reductase; a 3-hydroxypropionate dehydrogenase; a 3-hydroxypropionyl-CoA synthase; a CoA-transferase; a β-ketothiolase; a 3-hydroxyacyl-CoA dehydrogenase; a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, and a trans-2-enoyl-CoA reductase. 
     
     
         21 . The method of  claim 1 , wherein said method is performed in a recombinant organism. 
     
     
         22 . The method of  claim 21 , wherein said organism is subjected to a cultivation strategy under aerobic, anaerobic or, micro-aerobic cultivation conditions. 
     
     
         23 . The method of  claim 21 , wherein said organism is cultured under conditions of nutrient limitation. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 21 , wherein the principal carbon source fed to the fermentation derives from a biological feedstock. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 21 , wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock. 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 21 , wherein the organism is a prokaryote. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . The method of  claim 21 , wherein the organism is a eukaryote. 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 21 , wherein the organism's tolerance to high concentrations of a C7 building block is improved through continuous cultivation in a selective environment. 
     
     
         36 . The method of  claim 21 , wherein said host comprises an attenuation to one or more of the following enzymes: polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, NADH-consuming transhydrogenase, an NADH-specific glutamate dehydrogenase, a NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase, an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates, a butaryl-CoA dehydrogenase, or an adipyl-CoA synthetase. 
     
     
         37 . The method of  claim 21 , wherein said organism overexpresses one or more genes encoding: an acetyl-CoA synthetase; a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; a diamine transporter, a dicarboxylate transporter, and/or a multidrug transporter. 
     
     
         38 . A recombinant organism comprising at least one exogenous nucleic acid encoding (i) a β-ketothiolase, (ii) a thioesterase or a CoA transferase, and one or more of (iii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iv) an enoyl-CoA hydratase, and (v) a trans-2-enoyl-CoA reductase, said organism producing 7-hydroxyheptanoate. 
     
     
         39 . The recombinant organism of  claim 38 , said host further comprising one or more of the following exogenous enzymes: a monooxygenase, an alcohol dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxyvalerate dehydrogenase, a 6-hydroxyhextanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 5-oxovalerate dehydrogenase, or an aldehyde dehydrogenase, said host further producing pimelic acid. 
     
     
         40 . The recombinant organism of  claim 38 , said host further comprising one or more of the following exogenous enzymes: a monooxygenase, a transaminase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, and an alcohol dehydrogenase, said host further producing 7-aminoheptanoate. 
     
     
         41 . The recombinant organism of  claim 38 , said host further comprising one or more of the following exogenous enzymes: a carboxylate reductase, a ω-transaminase, a deacylase, an N-acetyl transferase, or an alcohol dehydrogenase, said host further producing heptamethylenediamine. 
     
     
         42 . The recombinant organism of  claim 38 , said host further comprising an exogenous carboxylate reductase and an exogenous alcohol dehydrogenase, said host further producing 1,7-heptanediol. 
     
     
         43 . The recombinant organism of  claim 38 , said host further comprising one or more of the following exogenous enzymes: an alpha-aminotransaminase; a 2-oxoadipate decarboxylase; a branch-chain decarboxylase; a glutamate decarboxylase; a ω-transaminase; a CoA-ligase; a CoA-transferase; and an alcohol dehydrogenase. 
     
     
         44 . (canceled) 
     
     
         45 . A nucleic acid construct or expression vector comprising
 (a) a polynucleotide encoding a polypeptide having β-ketothiolase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having β-ketothiolase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 1 or 13;   (b) a polynucleotide encoding a polypeptide having ω-transaminase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having ω-transaminase activity is selected from the group consisting of: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NOs: 7-12.   
     
     
         46 . A composition comprising the nucleic acid construct or expression vector of  claim 45 . 
     
     
         47 . A non-naturally occurring biochemical network comprising at least one substrate of  FIG. 1 , at least one exogenous nucleic acid encoding a polypeptide having the activity of at least one enzyme of  FIG. 1  and at least one product of  FIG. 1 . 
     
     
         48 . A bio-derived product, bio-based product or fermentation-derived product, wherein said product comprises:
 i. a composition comprising at least one bio-derived, bio-based or fermentation-derived compound according to  claim 1 , or of  FIG. 1  or any combination thereof,   ii. a bio-derived, bio-based or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of i., or any combination thereof,   iii. a bio-derived, bio-based or fermentation-derived resin comprising the bio-derived, bio-based or fermentation-derived compound or bio-derived, bio-based or fermentation-derived composition of i. or any combination thereof or the bio-derived, bio-based or fermentation-derived polymer of ii. or any combination thereof,   iv. a molded substance obtained by molding the bio-derived, bio-based or fermentation-derived polymer of ii. or the bio-derived, bio-based or fermentation-derived resin of iii., or any combination thereof,   v. a bio-derived, bio-based or fermentation-derived formulation comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., or bio-derived, bio-based or fermentation-derived molded substance of iv, or any combination thereof, or   vi. a bio-derived, bio-based or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based or fermentation-derived composition of i., bio-derived, bio-based or fermentation-derived compound of i., bio-derived, bio-based or fermentation-derived polymer of ii., bio-derived, bio-based or fermentation-derived resin of iii., bio-derived, bio-based or fermentation-derived formulation of v., or bio-derived, bio-based or fermentation-derived molded substance of iv., or any combination thereof.   
     
     
         49 . The method of  claim 14 , wherein said ω-transaminase has at least 70% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs.: 7-12.